Journal of Chromatography B, 836 (2006) 111115

Short communication

A demonstration of the use of ultra-performance liquid

chromatographymass spectrometry [UPLC/MS] in the determination
of amphetamine-type substances and ketamine for forensic
and toxicological analysis
Luigino G. Apollonio a, , Dennis J. Pianca b , Ian R. Whittall b ,
William A. Maher a , Jennelle M. Kyd a
a National Centre for Forensic Studies, University of Canberra, Bruce ACT 2601, Australia
b Department of Toxicology and Forensic Chemistry, ACT Government Analytical Laboratory, Weston Creek, ACT 2611, Australia
Received 9 March 2006; accepted 10 March 2006
Available online 17 April 2006

Abstract
We have recently seen the emergence of ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry as an alternative to
traditional high-performance liquid chromatography techniques. The strengths of UPLC technology promote the ability to separate and identify
drug compounds with significant gains in resolution and sensitivity and marked reductions in the overall time of analysis. As increased throughput is
the desire of the practical toxicology laboratory, the aim of this study was to trial commercially available technology by assessment of the separation
of several commonly encountered amphetamine-type substances. From injection of a poly-drug reference standard and whole blood extract, we
successfully separated and identified amphetamine, methamphetamine, ephedrine, pseudoephedrine, phentermine, MDA, MDMA, MDEA and
ketamine in less than 3 min using the Acquity UPLC-Micromass Quattro Micro API MS instrumentation (Waters Corporation, USA). In addition to
this significant reduction in overall run time, all peaks exhibited acceptable resolution using selected ion recording (SIR), with analysis indicating
the capability to separate 511 peaks in 1.75 min using the current system parameters. From this introductory data, it is therefore indicated that
the technological advancements defining ultra-performance liquid chromatography will allow it to serve as a powerful analytical tool for rapid
throughput analysis.
2006 Elsevier B.V. All rights reserved.

Keywords: Ultra-performance liquid chromatography; UPLC; Amphetamine-type substances; Mass spectrometry; Methamphetamine; MDMA; Ketamine

1. Introduction now promoted the widespread use of LC/MS as an analytical

It is only within the last decade that the use of traditional
high-performance liquid chromatographymass spectrometry
(HPLC/MS) has been readily accepted by the practical working
toxicology laboratory for high throughput analysis. Relatively
recent advances, notably the use of atmospheric pressure ion-
ization (API) techniques such as electrospray ionization (ESI)
and atmospheric pressure chemical ionization (APCI), have
Analyses performed at the testing laboratory of Waters Corporation,
Rydalmere NSW 2116, Australia.
Corresponding author. Tel.: +61 2 6201 5866; fax: +61 2 6201 2461.
E-mail address: Luigino.Apollonio@canberra.edu.au (L.G. Apollonio).
tool [1,2]. Since review articles by Maurer [3] and Marquet
and Lachatre [4], the number of publications directly rele-
vant to applications in forensic and clinical toxicology has
increased, mainly with a focus on improvements in separa-
tion and identification relative to gas chromatographymass
spectrometry (GC/MS). Hoja et al. noted that nearly 70% of
everyday samples encountered in toxicological laboratories can
be handled using LC [5], and the advantages of LC have been
well described, particularly the capabilities in analyzing polar
or thermolabile compounds without requiring derivatization
[69].
The use of LC/MS is now commonplace, therefore it
was only logical to push the technology closer to the the-
oretical capacities of the instrumental components. Much

1570-0232/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchromb.2006.03.045
112 L.G. Apollonio et al. / J. Chromatogr. B 836 (2006) 111115
more recently, the desire for significantly reduced analysis
times with increased sample throughput, sensitivity, and res-
olution has resulted in the development of ultrafast sepa-
rations and identification using LC/MS techniques [10,11].
Termed ultra-performance liquid chromatography (UPLC) or
ultrafast liquid chromatography, the improvements in such
parameters are largely due to advancements in the par-
ticle size and bridging structure of the column packing,
though complemented by additional instrumental modifications
[12,13].
The Acquity UPLC instrument (Waters Corporation, USA)
is an example of a commercially available UPLC system
with full instrument modifications: advancements in the sol-
vent delivery module, in combination with the column tech-
nology, allow the Acquity to run routinely at pressures up
to 15,000 psi; the Acquity sample manager has been modi-
fied to inject down to 1 l using a needle-in-needle probe;
and, the UPLC photodiode array and tunable UVvis detec-
tors have been modified accordingly in terms of increased
sampling rate and detector cell dispersion [14]. At the center
of the advancements, however, is the column, which utilizes
pressure-tolerant 1.7 m hybrid particles containing a bridged
ethylsiloxane/silica structure (BEH) [15]. It is the combination
of these technological developments that produce increased sen-
sitivities and improved peak resolution, while significant reduc-
tions in analysis times provide the rapid throughput desirable
to the working laboratory. Recent publications examining its
application in drug discovery and metabolism have been pro-
duced demonstrating the improvements in sensitivity, resolution,
and analysis time [1619], and have also included the sepa-
ration of the common Ephedra alkaloids ephedrine and pseu-
doephedrine [20]. However, few studies to date have examined
the separation of common and novel illicit amphetamine-type
substances and designer analogues often encountered in the
forensic laboratory in solid-state form or in biological speci-
mens.
Therefore, as this technology should be of significant practi-
cal advantage to the forensic/post-mortem toxicology labora-
tory, we initially tested the system with a poly-drug refer-
ence standard containing eight amphetamine-type substances:
amphetamine, methamphetamine, 3,4-methylenedioxyamphe-
tamine (MDA), 3,4-methylenedioxymethamphetamine (MD-
MA), 3,4-methylenedioxyethamphetamine (MDEA), ephed-
rine, pseudoephedrine, and phentermine. This poly-drug stan-
dard also contained phenylethylamine, a commonly encountered
putrefactive amine indicative of decomposition [2124]. In addi-
tion to the standard mix, we also evaluated a whole blood
extract that was reflective of the high toxic range observed
in forensic/post-mortem toxicology. This sample contained
amphetamine, methamphetamine, MDA, MDMA, phenylethy-
lamine, and ketamine, the latter of which was included due to
its increasing occurrence in club- or designer drug street sam-
ples [25]. From analysis of these two reference solutions, we
provide a demonstration of the new UPLC technology and gain
an indication of any potential in improvements in analysis time,
peak separation, and identification power when coupled to mass
spectrometry.
2. Experimental
Amphetamine, methamphetamine, MDA, MDMA, MDEA,
ephedrine, pseudoephedrine, phentermine, and phenylethy-
lamine (hydrochloride salts) were obtained from the National
Measurement Institute (Sydney, NSW), and a methanolic
stock solution was prepared in a concentration of 10 g/mL.
Four microlitres of this stock was then injected on the
Acquity system (approximating 40 ng of each drug of interest
injected). In addition, a whole blood sample was spiked with
amphetamine, methamphetamine, MDA, MDMA, phenylethy-
lamine, and ketamine for a final concentration of 2 g/mL.
Extraction of this sample was then performed using an Oasis
MCX solid phase extraction column (60 mg, Waters Corpo-
ration, USA) by the following procedure. Two millilitres of
100 mM sodium phosphate buffer (pH 6) was added to the
1 mL of spiked blood, followed by vortex-mixing and cen-
trifugation. The column was conditioned with 3 mL methanol,
3 mL deionized water, and 1 mL phosphate buffer (pH 6).
The sample was introduced to the column, allowed to pass,
then followed by 3 mL deionized water, 1 mL 0.1 M acetic
acid, and 3 mL methanol. The column was then dried under
vacuum at approximately 5 psi for 10 min, then elution per-
formed using two 1 mL fractions of 10% ammonium hydrox-
ide in methanol. Five microlitres of this was injected on the
Acquity system. No concentration step was performed, lead-
ing to approximately 5 ng of each drug injected assuming 100%
recovery.
2.1. UPLC conditions
The column used was Waters UPLC BEH C18 (2.1 mm
50 mm), with the target temperature set at 40 C. The
Waters Acquity TUV single wavelength detector was
programmed for analysis at 254 nm. The mobile phase
used for the Waters Acquity UPLC-UV and MS system
was aqueous pyrrolidine (0.5 mL glacial acetic acid and
1.0 mL pyrrolidine in 500 mL reagent grade water) and
methanol under isocratic conditions (flow rate 0.4 mL/min;
50:50 for methanolic standard; 52:48 for whole blood
extract).
2.2. MS conditions
A Waters Micromass Quattro Micro API mass spec-
trometer instrument (data analysis software MassLynx V4.0)
was used in positive electrospray ionization mode. Nitro-
gen was used as the drying gas. Desolvation gas flow was
648 L/h, and cone gas flow was maintained at 58 L/h. Des-
olvation temperature was 445 C and source temperature was
119 C. Observed capillary and cone voltages were 3510 V and
22.83 V, respectively (note: improved response was observed
for MDA using a cone voltage of 50 V, therefore this param-
eter was included for MDA in the range for the whole blood
extraction analysis program; similarly, 15 V was used for
amphetamine).
 L.G. Apollonio et al. / J. Chromatogr. B 836 (2006) 111115 113

Fig. 1. (A) Selected ion recording (SIR) chromatograms of a methanolic drug poly-standard containing eight amphetamine-type substances with phenylethylamine
putrefactive marker ([M + H]+ ions monitored are in right hand margin; isocratic 50:50 aq. pyrrolidine:methanol at 0.4 mL/min); (B) Selected ion recording (SIR)
chromatograms of four amphetamine-type substances, phenylethylamine, and ketamine from extracted whole blood (baseline at zero, non-linked vertical axes;
isocratic 52:48 aq. pyrrolidine:methanol at 0.4 mL/min).
114 L.G. Apollonio et al. / J. Chromatogr. B 836 (2006) 111115
3. Results and discussion
The injection of the polydrug reference standard showed
favorable separation and acceptable peak resolution with regard
to the concentration injected (Fig. 1A), and it was observed that
all eight amphetamine-type substances separated in less than
2.5 min under the 50:50 isocratic conditions. While analytical
methods using MS and MS/MS have been described for such
substances with individual peak retention times ranging from
1.5 min to 13 min [6,8,9,26] none of these exhibit both the peak
separation and total analysis time we observed from a drug mix-
ture by both tunable UV detection and selected ion recording
(SIR). This is strong indication of the potential of UPLC to
significantly reduce analysis time while promoting baseline sep-
aration for these licit/prohibited drug compounds.
The individual SIRs for each of the eight substances are
shown in Fig. 1A, in addition to that of the decomposition
marker, phenylethylamine. Note that the protonated molecular
ions ([M + H]+ ) monitored are indicated along the right mar-
gin of the SIR chromatograms. From the elution order and
approximate peak-to-peak resolution (Table 1), the observed
potential for coelution interference appears minimal under
the applied instrument conditions and sample concentration.
Approximate baseline peak-to-peak resolution was calculated
from R = (trb tra )/((1/2)(Wba + Wbb )), where tr is retention time
and Wb is peak width at base; the value of 1.0 indicates 10%
overlap, and 1.5 illustrates complete resolution of two peaks of
equal size [27]. From our results (Table 1), the resolution of the
phenylethylamine putrefactive base (0.87 min) indicates poten-
tial concentration-dependent coelution with either ephedrine
(0.79 min) or pseudoephedrine (0.91 min), but it is unlikely this
would interfere with mass spectral identification in a forensic
toxicological case with the use of selected ion recording. Sim-
ilarly, there is partial coelution of phentermine (1.72 min) and
methamphetamine (1.81 min) observable at m/z 150. This poten-
tial quantitation interference could be alleviated with the use of
an optimized gradient in the circumstance that these two com-
pounds are encountered in a toxicological specimen.
Table 1
Retention times and peak-to-peak (PtP) resolution of ephedrine, phenylethylamine, pseudoephedrine, MDA, amphetamine, MDMA, phentermine, methamphetamine,
MDEA, and ketamine (by elution order)
Peak Retention time (min)
Methanolic poly-drug standard (isocratic 50:50 aq. pyrrolidine:methanol at 0.4 mL/min)
Ephedrine 0.79
Phenylethylamine 0.87
Pseudoephedrine 0.91
MDA 1.07
Amphetamine 1.20
MDMA 1.56
Phentermine 1.72
Methamphetamine 1.81
Whole blood extract (isocratic 52:48 aq. pyrrolidine:methanol at 0.4 mL/min)
Phenylethylamine 0.93
MDA 1.18
Amphetamine 1.35
MDMA 1.77
Methamphetamine 2.04
In the analysis of the extracted whole blood sample (Fig. 1B),
the change in isocratic conditions (from 50% to 52% aq. pyrro-
lidine) caused an expected shift in the separation of the five
compounds (phenylethylamine, MDA, amphetamine, MDMA,
methamphetamine). Again, there was favorable separation and
resolution taking into account concentration considerations, as
indicated by the SIRs (Table 1, Fig. 1B). Note that ketamine
is observable in less than 2.5 min as well. Thus, while there is
potential for ketamine to coelute with MDEA when using UV
detection, there are markedly discernible fragmentation patterns
in the mass spectral data that would allow positive identifica-
tion and quantitation of both. Should coelution of ketamine and
MDEA be observed in a toxicological or forensic chemistry case
utilizing only UV detection (e.g. at 254 nm), altering the iso-
cratic conditions or employing an optimized gradient too would
likely correct this. Also, it should be noted that a major fragment
from ketamine occurs at m/z 180, the ion used for the identifica-
tion and quantitation of MDA. This is observable as the peak at
2.37 min in the MDA SIR of Fig. 1B. From the separation using
the stated conditions, this ketamine artefact does not interfere
with the identification of MDA in any way, but could possibly be
used as a secondary confirmation for the presence of ketamine.
Therefore, from the data for the extracted whole blood speci-
men, the UPLC conditions again indicate favorable separation,
sensitivity, and resolution when challenged with the task of tox-
icological interpretations of therapeutic to toxic drug levels.
With peak widths (Wp ) ranging from 0.1 min to 0.2 min in
both the methanolic poly-drug standard and the whole blood
extract, the improved resolving power of UPLC is evident in
reference to the total run time. The initial peak to final peak
analysis time in the case of the standard solution is 1.75 min,
providing a separation range of 511 peaks based on a res-
olution factor of 1.5 for complete baseline separation (calcu-
lated as 1.75/(1.5 WP )). Resolving power for the whole blood
extract is similar at 710 peaks in 1.60 min. One could then
imagine the further power of the ultra-performance instrumen-
tation when considering adjustments to column length and run
time.
Next peak Approximate PtP resolution
Phenylethylamine 1.0
Pseudoephedrine 0.5
MDA 2.1
Amphetamine 1.7
MDMA 3.6
Phentermine 2.1
Methamphetamine 1.2
MDEA (2.38 min) 3.8
MDA 3.3
Amphetamine 2.2
MDMA 5.6
Methamphetamine 2.7
Ketamine (2.38 min) 3.4
 L.G. Apollonio et al. / J. Chromatogr. B 836 (2006) 111115
4. Conclusion
These introductory trials of the ultra-performance technology
have demonstrated the potential of rapid separation and identi-
fication of illicit drugs such as amphetamine-type substances.
While more research and system optimization are required
before reporting the full validation of the instrumental method,
analysis has indicated prospective gains in sensitivity and res-
olution, with a strong illustration of the reduction in time of
analysis. The ability to separate and identify eight amphetamine-
type substances and ketamine in less than 3 min with acceptable
baseline resolution is a powerful tool providing opportunity to
increase the throughput of the working toxicology laboratory
without sacrificing the quality of the analysis.
Acknowledgements
The authors wish to thank Henry Chan, Brian Walker, and
Steve Wilson of Waters Corporation and Dr. Ian McNaught of the
University of Canberra for their professional advice and assis-
tance.
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